Table 1.

Outline summary, including capabilities, limitations and key experimental variables, of the principal physical methods for the investigation of structural colour in biological systems.

technique	capabilities	limitations	key experimental variables
spectrophotometry (including microspectrophotometry and bifurcated fibre probe use)	used for measuring optical scattering from or through samples; polarized or unpolarized illumination can be provided with broadband or laser sources and appropriate filters; sample orientation and light incidence angle can usually be controlled; light can be delivered using optical fibres or conventional lenses; detection of scattered light achieved using fibre-fed spectrometers (for white light illumination) or large-area photodiodes (for laser or monochromatic light illumination)	difficult to measure absolute values of reflectance or transmittance; great care should be taken when using white reference standards; use of microspectrophotometer or bifurcated fibre probe precludes independent variation of angles of light incidence and scatter detection	incident and scattered light wavelengths; angle of incidence; scattered light detection angle; sample orientation; incident and scattered light polarization state; incident and scattered light intensities
integrating sphere photometry	used for measuring optical scattering from or through samples; light delivered using optical fibres or conventional lenses (dependent on sphere design); detection of scattered light achieved using fibre-fed spectrometers (for white light illumination) or large-area photodiodes (for laser or monochromatic light illumination; dependent on sphere design)	sample orientation and the illumination and scattered light detection angles are not selectable; there is no polarization selectivity; great care should be taken when using white reference standards	incident and scattered light wavelengths; incident and scattered light intensities
scatterometry and reflectometry	used for measuring scattered light distribution by applying point source illumination, the direction of which can be varied; detection can be realized with scanning fibres connected to a photodiode array spectrophotometer or by imaging	measurements with scanning fibres are extremely laborious; imaging methods require special optical methods when large spatial angles are to be spanned	the measured bidirectional reflectance distribution function involves knowledge of spatial directions of incident and scattered light, wavelength, polarization
scaled model fabrication and electromagnetic interrogation	used for replicating accurate millimetre- and centimetre-scale models appropriate for interrogation using much longer wavelengths (e.g. microwaves); replication processes are variable, but can include rapid prototyping and laser sintering; large-scale models are much easier to position and manipulate accurately; for biomimetic application studies, the models may be fabricated using one or more of a wide range of dielectric, semiconductor or metallic materials	the material used to replicate large-scale models should have analogous optical properties to the original optical sample	incident and scattered light wavelengths; angle of incidence; scattered light detection angle; sample orientation; incident and scattered light polarization state; incident and scattered light intensities
SEM	used for imaging the surfaces or exposed interiors of samples; typical available resolutions are a few nanometres to tens of nanometres; samples may be pre-prepared with an FIB procedure, freeze–fracture or mechanically induced damage to reveal subsurface structures; estimated dimensions of samples' surface nanostructure	owing to two-dimensional perception issues, SEM offers much less accuracy than TEM in quantifying dimensions of subsurface structures; poor sample preparation may lead to low image quality owing to electrical charging issues	orientation of the sample on the SEM stub; metal overlayer thickness; sample stub orientation in SEM; working distance; beam current and voltage; SEM detector mode (e.g. backscattered, secondary electrons)
TEM	used for imaging ultramicrotomed samples' thin (approx. 70 nm thick) sections; typical available resolutions are a few nanometres to tens of nanometres; different staining processes create differential greyscale contrast in the sample's nanostructure; accurate quantification of nanostructured dimensions and structural geometries is possible	ultramicrotome facilities and expertise with biophotonic sample sectioning have limited availability; poor sample fixing and staining protocols, in addition to poor sectioning technique may lead to artificial results	sample preparation fixative, impregnation and staining chemicals; ultramicrotome section thicknesses and orientations; TEM E-beam spot diameter and voltage
AFM	used for forming digital maps of samples' surface features; enables estimates of large area surface height distributions, which is not accurately possible with either SEM or TEM	does not work accurately for samples that exhibit features with significant height-to-separation ratios, or with crevice-like or partially covered subsurface features	operating mode (e.g. contact mode, tapping mode, etc.); probe-tip profile